[PMC free article] [PubMed] [Google Scholar] 18. assay reagents, such as dithionitrobenzene. Through the use of a combination of wild-type (WT) GlmU and a variant lacking acetyltransferase activity (GlmUH363N), we report here a robust coupled assay CENPA to screen for inhibitors specific to the acetyltransferase reaction of the GlmU enzyme. Using this assay, a total of 63 molecules, 37 specific to the acetyltransferase activity of GlmU, were selected for further study. NMDA The potency and specificity of three identified small-molecule inhibitors highlight them as excellent candidates for further study. MATERIALS AND METHODS Materials. 4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), DTT, magnesium chloride (MgCl2), and AcCoA were purchased from BioShop Canada Inc. (Burlington, Ontario, Canada). Library compounds were purchased from Maybridge (Cornwall, United Kingdom). All other chemicals were purchased from Sigma-Aldrich (Oakville, Ontario, Canada). Protein expression and purification. Native GlmU was purified as previously described (19). Briefly, a pET3a expression vector harboring the gene was transformed into BL21(DE3) which massively expressed NMDA the enzyme in the absence of induction. After streptomycin sulfate precipitation of DNA and centrifugation, the supernatant was applied to a Poros HQ50 anion-exchange column and eluted with an NaCl gradient (0 to 0.75 M). The fractions were analyzed by polyacrylamide gel electrophoresis, pooled, and concentrated for separation using Superdex 200 prep grade gel filtration chromatography. The apparent purity of the enzyme was 98% as judged by Coomassie blue-stained polyacrylamide gel electrophoresis. The GlmUH363N mutant enzyme was overexpressed and purified using the same protocol. Coupled acetyltransferase assay. The acetyltransferase activity of GlmU was monitored in a coupled assay with the end product inorganic phosphate detected using malachite green (4) as described here. The formation of GlcNAc-1-P (are the standard deviations () and the averages () of the high (is percent activity, is minimum %is maximum %is the concentration of inhibitor, and is the slope factor: (2) Each primary screen hit was tested for nonspecific inhibition by the addition of 0.01 mg/ml bovine serum albumin (BSA) to the primary screen reaction (14). Compounds (at 10 M) were tested for inhibition of the acetyltransferase activity in the presence of the BSA protein. Mode-of-inhibition studies. Mode-of-inhibition and values NMDA were determined by simultaneously changing the inhibitor concentration and either the AcCoA or GlcN-1-P concentration (12.5 M to 200 M). The resulting curves were fit by nonlinear least-squares regression to models describing competitive, uncompetitive, or mixed inhibition (25) using the Enzyme Kinetics Module 1.1 of Sigmaplot 8.0 (SPSS Inc., Chicago, IL). RESULTS Suitability of the developed assay to high-throughput screening. To predispose the developed assay to inhibitors of the acetyltransferase domain, the acetyltransferase reaction was maintained as rate limiting. With the addition of 100-fold excess of GlmUH363N (25 nM) over WT GlmU (0.25 nM), the rate of the reaction was increased 3.5-fold, consistent with the rate differences between the two enzymatic reactions (21 s?1 for uridylyltransferase and 77 s?1 for acetyltransferase ). The ratio of mutant enzyme to WT enzyme was saturating for uridyltransferase activity, while maintaining GlmUH363N at a low concentration to prevent appreciable binding of the tested compounds to the variant. An excess of inorganic pyrophosphatase ensured efficient cleavage of pyrophosphate to maintain the acetyltransferase activity as rate limiting. The developed assay was found to be linear with both time and the concentration of WT enzyme. A schematic outlining the reaction is displayed in Fig. ?Fig.1A1A. Open in a separate window FIG. 1. (A) Scheme of the developed coupled acetyltransferase assay for GlmU. The addition of excess GlmUH363N and inorganic pyrophosphatase (PPase) have rendered the acetyltransferase activity rate limiting. The 2 2 units of inorganic phosphate produced by each acetyltransferase turnover are detected by absorbance of 660 nm after the addition of malachite green assay reagents. (B) Evaluation of GlmU acetyltransferase screen. The high controls (100% residual activity; circles) and the low controls (0% residual activity; squares) are plotted. The assay shows a large hit window between the high and low controls with a calculated Z factor of 0.71. The statistical parameter GlmU acetyltransferase reaction. The percent residual activity (%RA) of duplicates is plotted on opposite axes. Data points that fall on a slope of 1 1 are considered to be in agreement. Molecules within the outlined area below 60% residual activity for each replicate reproducibly showed inhibition below the determined cutoff value and were selected for further study. Specificity of inhibitors to GlmU’s acetyltransferase domain. Each hit molecule from the.